MX2010009430A - Oligosaccharide ingredient. - Google Patents

Abstract

An oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula RnSacm where R is an amino acid residue, Sac is a monosaccharide selected from the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has a value between (1) and (10) with the proviso that if n has the value (1) R is a threonine residue or a serine residue and if n has a value between (2) and (10) the peptide contains at least one threonine or serine residue, m has a value between (2) and (4) and at least (20) mol% of the ingredient is N-acetyl-neuraminic acid.

Description

OLIGOSÁCARIDO INGREDIENT Field of the invention The invention relates to an oligosaccharide ingredient rich in sialic acid, food products comprising said oligosaccharide ingredient and processes I for producing said oligosaccharide ingredient. j BACKGROUND OF THE INVENTION It is known that human milk contains a large amount of nondigestible oligosaccharides indigestible. In fact, nondigestible oligosaccharides represent the third largest solid component (after lactose and lipids) in breast milk, occurring at a concentration of 12-15 g / l in colostrum and 5-8 g / l in mature milk The oligosaccharides of human milk are very resistant to enzymatic hydrolysis, indicating that these oligosaccharides can exhibit essential functions not directly related to their calorific value. ! During the past two decades, the chemical structures of human milk oligosaccharides have been studied using NMR spectroscopy and mass spectrometry. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine with many and varied links between them, thus accounting for the enormous number of different oligosaccharides in the human milk - more than 130 of these structures have been identified so far. Almost all of them have a portion of lactose in its extreme redactor whereas the sialic acid (when present) occupies the terminal position in the eitremo no reducer. Of particular interest is the large amount of sialylated oligosaccharides in human milk. Sialic acid is a nine-carbon sugar that is a vital structural and functional component of brain gangliosides. It is considered to play an essential role in nerve cell transmission, memory formation and cell-to-cell communication. Studies in rat pups indicate that I early supplementation with sialic acid improves both gangiioside sialic acid and learning ability in well nourished and malnourished animals and that these changes persist in the adult stage. Breast milk is recommended for all infants. However, in certain cases, breastfeeding is inadequate or unsuccessful for medical reasons, or the mother chooses not to breastfeed. For these situations the infant formulas have been developed. However, bovine milk that is usually used1 as free acetylneuraminic, sialylactose and other sialylated oligosaccharides, gangliosides containing sialic acid, and the casein glycomacropeptide peptide. The caseinoglycomacropeptide or CGMP is a hydrophilic peptide, rich in carbohydrate, which is the C-terminal portion of? casein from which it can be divided by enzymatic or acid treatment. In the United States Patent Application No. i 2005/0096295 is proposed to increase the sialic acid content of infant formula by including a novel CGMP ingredient having an improved concentration of sialic acid. This ingredient has a sialic acid content of more than 60 mg / g of peptide compared to 40 to 60 mg / g of peptide in CGMP of natural origin. It can be produced by fractionation using anion chromatography or by partial hydrolysis followed by fractionation using anion chromatography. However, there is still a need to provide other ingredients in sialic acid that can be used to enrich infant formula and other nutritional compositions in this important component.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides an oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula RnSacm wherein Rj is an amino acid residue, Sac is a monosaccharide selected from the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has a value between 1 and 10 with the proviso that if n has the value 1 R is a residual trace or! a serine residue and if n has a value between 2 and 10 the peptide contains at least one threonine or serine residue, m has a value between 2 and 4 and at least 20 mo% of the ingredient is N-acetyl-neuraminic acid. This ingredient is a new food-grade ingredient rich in sialic acid which is suitable for addition to nutritional compositions such as infant formula to increase the sialic acid content of the composition. The invention further extends to a process for producing an oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula i RnSacm wherein R is an amino acid residue, Sac is a monosaccharide selected from From the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and I galactose, n has a value between 1 and 10 with the proviso that if n has the value 1 R is a threonine residue or a serine residue if n has a value between 2 and 10 the peptide contains at least one threonine or serine residue, m has a value between 2 and 4 and at least 20 mol% of the ingredient is N-acetyl-neuraminic acid, which comprises the steps of hydrolyzing caseinoglycomacropeptide with an exoprotease and an endoprotease to obtain a mixture of free amino acids and peptides with a chain length between 2 and 10 and subject the hydrolyzed mixture to nanofiltration to retain the fraction having a molecular weight between 1000 and 2000 Daltons. The invention also extends to a food product comprising an oligosaccharide ingredient as described above. Optionally the food product is a product or infant formula, although the product can be any food or drink consumed by infants, infants or adults. The consumption of a food product containing said oligosaccharide ingredient can improve the definition that must be taken into account when reading and interpreting the description, examples and claims. j I "CGMP": casein-glycomacropeptide, that is, the glycomacropeptide that is divided from bovine? -sinin by means of the action of the renin enzyme.; i "Infant formula": food destined for the complete nutrition of raffles during the first four to six months of life and for his partial nutrition ':! later. The term includes both initial and follow-up formulas. I "Sialic acid": N-acetylneuraminic acid. The invention provides an oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula RnSacm wherein R is an amino acid residue, Sac is a monosaccharide selected from the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has a value between 1 and 10 with the proviso that if n has the value 1 R is a threonine residue or a serine residue and if n has a value between 2 and 10 the peptide contains a residue threonine or serine, m has a value between 2 and 4 and at least 20 moi% of the ingredient is N-acetyl-neuraminic acid as well as infant or adult food products comprising said oligosaccharide ingredient. j Preferably n has a value between 1 and 3 and m has a value of 3 or 4., | The ingredient contains at least 20 mol% sialic acid as part of a saccharide chain linked to the hydroxyl group of threonine or serine. The sialic acid can be part of the chain or can be a substitute for a monosaccharide unit in the chain. Thus, the oligosaccharide ingredient may contain the following monosaccharide: The oligosaccharide ingredient may contain from 15 to 25 mol% of a Serine and threonine mixture. The oligosaccharide ingredient may contain the following glycosylated amino acids or peptides: NeuAc-ot-2,3-Gal ^ -1, 3- (NeuAc-a-2,6 -) - GalNAc-Rn! NeuAc-a-2,3-Gal ^ -1, 3-GalNAc-Rn ¡Gal ^ -1, 3- (NeuAc-a-2,6 -) - GalNAc-Rn Gal- -1, 3-GalNAc-Rn The oligosaccharide ingredient of the invention can be produced by i hydrolysis of CGMP using an exoprotease and an endoprotease to obtain a mixture of free amino acids and peptides with a chain length between 2 and 10 and subjecting the hydrolysed mixture to nanofiltration to retain the fraction that has a molecular weight between 1000 and 2000 Daltons. j CGMP is a by-product of cheese making in which whole milk is treated with the renin enzyme to precipitate casein. In this process, CGMP is divided from? -casein and is maintained in solution with serum proteins. This product is known as sweet whey. The CGMP can be separated from whey proteins through any process known in the art. A suitable process is described in European Patent No. 986312. The hydrolysis can be carried out using any combination of enzymes. An example of a commercially available enzyme system with exo- and endo-protease activity is the product sold as Flavourzyme® by the Novozymes division of Novo Nordisk. The product of the hydrolysis process is a mixture of free amino acids, tréonina and glycosylated serine, glycosylated peptides containing at least one threonine or serine residue and non-glycosylated peptides, the peptides having a chain from 2 to 10 amino acid residues. This mixture is subjected to nanofiltration to separate and retain the fraction that has a molecular weight in the range from 1000 to 2000.

Daltons. This fraction will comprise the oligosaccharide ingredient of the invention. In a preferred aspect of the invention, the oligosaccharide ingredient described above is incorporated into a food product. In the context of the present invention, the term "food product" is intended to comprise any consumable matter. Therefore, it can be a product intended for consumption by humans, in particular infant formula, follow-up formula, baby food such as infant cereals and the like. In particular, the oligosaccharide ingredient of the invention can be incorporated into infant formulas, milk for growth and dehydrated milk or cereal mixtures. The food product can be prepared in any suitable manner known in the art according to the type of product and the oligosaccharide ingredient of the invention can be added to the product in an appropriate stage in the manufacturing process. For example, a infant formula can be prepared by combining together the source of protein, any carbohydrates other than lactose, and the source of fat in adequate proportions. It is possible to add emulsifiers if! you want At this point, vitamins and minerals can be added, although they are usually added later in order to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like can be dissolved in the source of fat before the combination. Water can then be mixed, preferably water that has been subjected to reverse osmosis, to form a liquid mixture. The liquid mixture can then be heat treated to reduce the bacterial loads. For example, the liquid mixture can be rapidly heated to a temperature in the range of about 80 ° C to about 10 ° C for about 5 seconds to about 5 minutes. This can be carried out by steam injection or by means of a heat exchanger, for example a plate heat exchanger. ! The liquid mixture can then be cooled to about 60 ° C or I to about 85 ° C, for example by instantaneous cooling. The liquid mixture can then be homogenized, for example in two stages at about 7 MPa to about 40 MPa in the first stage and about 2 MPa to about 14 MPa in the second stage. The homogenized mixture can then be further cooled to add any heat sensitive components such as vitamins and minerals. The pH and solids content of the homogenized mixture are conveniently standardized at this point. The homogenized mixture is transferred to a suitable dehydration apparatus, such as a spray drier, and converted to powder. The powder will have a moisture content of less than about 5% by weight. The oligosaccharide ingredient of the invention can be added directly to the infant formula by dry mixing or, if it is for liquid, in the combination diet described above. In both cases, it will be appreciated that the addition of the ingredient || i entails the simultaneous addition of an amount of amino-nitrogen and therefore it may be necessary to adjust the protein content of the infant formula or other product to which it is adding the ingredient consistently as it will be obvious to someone with experience in the art. The permissible protein content and the amino acid profile of infant formulas are specified in the legislation such as the European Commission Directive 91/321 / EEC of 14 May 1991 and the person with experience in the art will be able to easily adjust the protein and amino acid profile content of infant formula to consider the additional amino-nitrogen introduced by the ingredient of the invention as well as the additional content of threonine and serine. ! The final concentration of the oligosaccharide ingredient in the baby or infant formula or food product can be from 0.2 to 4.0%, preferably 0.5 to 2.0% by weight of dehydrated material. However, these amounts will not be considered as limiting and will be adapted for the target population, for example based on the weight and age or health status of the infant or infant. Preferably, the formula or food containing the oligosaccharide ingredient of the invention is supplied to the baby in each food. Although it is preferred to supplement food products specifically intended for the nutrition of infants or babies, it may be beneficial to supplement food products not specifically destined for or intended for the adult population. For example, the oligosaccharide ingredient of the invention can be incorporated into health care nutrition products and nutritional products for the elderly.

Said food products may include milk, yogurt, cottage cheese, cheese, fermented milks, fermented products based on milk, ice cream, fermented cereal products in Jase, or products based on milk, among others. In addition to the oligosaccharide ingredient of the invention, a infant food product such as a formula can comprise one or more additional oligosaccharides that are added separately. The invention will now be illustrated by reference to the following example.

Example 1 j The following is an example of the composition of a infant formula containing an oligosaccharide ingredient according to the present invention.

Nutrient per 100 kcal liter Energy (kcal) 100 670 Protein (g) 1.83 12.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 A-linolenic acid (mg) 101 675 Lactose (g) 11.2 74.7 Ingredient OS (g ) 0.15 1.0 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (pg) 8 50 Se (pg) 2 13 Vitamin A RE) 105 700 Vitamin D (g) 1.5 10 Vitamin E (mg TE) 0.8 5.4 Vitamin K1 (ug) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Phosphoric acid (pg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (pg) 0.3 2 Biotin (pg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 15 100 Cu (mg) 0.06 0.4 Zn (mg) 0.75 5 Example 2 j I 50g of caseinoglycomacropeptide (Biopure GMP, Davisco, USA) were dissolved in 10% (w / v) in 500ml of deionized water. The solution heated at 40 ° C for 30 minutes with moderate agitation and recirculated through a tangential laboratory flow filtration system (Millipore, USA) with a Pellicon filter cartridge 10O0 Da (0.1 m2) at a feed pressure 1.2 bar and without holding pressure. J Subsequently, an aminopeptidase from Aspergillus oryzae was added (Flavourzyme 1000L containing 1000 aminopeptidase units per gram (l_JpU / g), Novozymes, DK) at 3500 LAPU per 50g of protein. After about 1 hour, diafiltration was started by increasing the retentate pressure to 3 bars with a feed pressure of 3 bars. After 5 hours the filtration was stopped and the retentate was collected. The retentate and the 7 permeate fractions that were harvested during the process were lyophilized. Then, the content of dehydrated matter (DM) and sialic acid of each fraction was determined. The acid content was measured sialic after moderate acid hydrolysis and labeled using the DMB method (1,2-diamino-4,5-methylenedioxybenzene dihydrochloride) and analysis on an HPLC equipped with a Shodex C18 reversed phase column and a fluorescence detector Quej uses Authentic NeuAc as external standards. Table 1 shows the balance of mass and i the content of sialic acid in the starting material, permeate and retentate fractions.

Table 1 mass (g) NeuAc (% of D) yield (%) enrichment í starting material 50 7.5 100 LOO 'I permeate 1 4.32 0.2 8.64 0.03 permeate 2 3.1 0.2 6.2 0.02 permeate 3 6.1 0.3 12.2 0.04 permeate 4 4.29 1.4 8.58 0.18 permeate 5 2.8 0.4 5.6 0.05 I permeate 6 2.8 0.4 5.6 0.05 permeate 7 2.94 0.3 5.88 0.04 retained 22.6 14.4 45.2 1.92 The content of galactose and N-acetyl-galactosamine in the retentate was determined after acid hydrolysis using an HPAEC equipped with a Dionex CarboPac PA1 analytical column and a driven amperimetry detector. Briefly, the retentate was dissolved in water and tri-fluoroacetic acid was added to a final concentration of 2M. The solution was heated for 3 hours at 100 ° C, dehydrated under an N-stream and dissolved again with water. After filtering through a 22 μm particle filter, the sample was analyzed. Quantification with authentic galactose and galactosamine (N-acetylgalactosamine converted to galactosamine during acid treatment) was performed as external standards. The final dehydrated retentate contained 24.4% by weight of (NeuAc) 2-Gal- GalNAc-Thr with the similar structure NeuAc-a-2,3-Gal-p-1, 3- (NeuAc-2,6 -) - GalNAc threonine / peptide with 43 mol% sialic acid, 20.5 mol% of galactose, 18.4% mol N-acetyl galactosamine and 18 mol% threonine. !

Claims (4)

? CLAIMS

1. An oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula RnSacm characterized in that R is a residue of; Amino acid, Sac is a monosaccharide selected from the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has a value between 1 and II 10 with the proviso that if n has the value 1 R is a threonine residue or a serine residue and if n has a value between 2 and 10 the peptide contains at least one residue threonine or serine, m has a value between 2 and 4 and at least 20% mole of the ingredient is N-acid serine or a mixture thereof. ! 3. An oligosaccharide ingredient according to claim 1 or 2, further characterized in that n has a value between 1 and 3. 4. An oligosaccharide ingredient according to any preceding claim, further characterized in that m has a value of

2. or

3. A food product comprising an oligosaccharide ingredient according to any of claims 1 to

4. I 6. A food product according to claim 5, which is a infant formula. 7. A food product according to claim 5 or 6, which comprises from 0.2 to 4.0% by weight of the oligosaccharide ingredient. | 8. A process for producing an oligosaccharide ingredient in accordance with any of claims 1 to 4 comprising the steps of hydrolyzing caseinoglycomacropeptide with an exoprotease and an endoprotease to obtain a mixture of free amino acids and peptides with a chain length between 2 and 10 and I subjecting the hydrolysed mixture to nanofiltration in order to retain the fraction that has a molecular weight between 1000 and 2000 Daltons